Neutrophil Oxidative Burst Activity Research Articles

The influence of probiotic administration on the phagocytic and oxidative burst activity of neutrophils and monocytes in the peripheral blood of dairy cows during different lactation periods

Abstract Introduction The objective of this bovine peripheral blood study was a comparative assessment of the phagocytic activity of neutrophils and monocytes and of the intracellular killing capacity of neutrophils from cows given no probiotic and from cows which were administered a probiotic consisting of Saccharomyces cerevisiae, Lactobacillus acidophilus, Lactobacillus plantarum and Rhodopseudomonas palustris. These activity types were compared during different lactation periods. Material and Methods A cohort of 20 pregnant dairy cows was divided into two groups of 10. The experimental group consisted of cows fed a ration supplemented with probiotics, and the control group consisted of cows fed an unsupplemented ration. Blood was drawn six times: 7 days before dry off, 14 days before parturition, and 7, 21, 60 and 90 days postpartum (DPP). The phagocytic activity of neutrophils and monocytes and the oxidative burst activity of neutrophils were determined by flow cytometry in the peripheral blood of all examined cows. Results Phagocytosis testing revealed increased percentages of phagocytic neutrophils and monocytes in the experimental group at 21, 60 and 90 DPP (P-value < 0.01). The mean fluorescence intensity (MFI) values for neutrophils and monocytes were higher on all days of the study (P-value < 0.01). In oxidative burst testing, the percentages of detected neutrophils and their MFI were increased in the experimental group on all days (P-value < 0.01). Conclusion The use of probiotics supported dairy cows’ immunity throughout the whole experiment. Probiotic supplementation may limit the occurrence of infectious diseases in these animals.

Lupus nephritis with clinical and laboratory features of humoral immune deficiency in a 29-year-old female with a novel NCF1 variant consistent with autosomal recessive chronic granulomatous disease

Chronic Granulomatous Disease (CGD) is a rare, inherited disorder of the innate immune system characterized by defects in the phagocyte NADPH oxidase complex. This results in poor phagocyte function causing frequent infections (bacterial and fungal) and chronic inflammation. Here we discuss a rare presentation of lupus nephritis and antibody deficiency in an adult female patient with a novel homozygous nonsense mutation in the NCF-1 gene causing autosomal recessive CGD.Our patient is a 29-year-old female with history of ITP, biopsy-confirmed lupus nephritis, systemic lupus erythematosus (SLE), idiopathic avascular necrosis of the hip, and previous diagnosis of common variable immune deficiency (CVID) treated with Immunoglobulin replacement therapy (IVIG). She presented to our clinic to establish care in the setting of persistent skin abscesses/cellulitis, poor wound healing, necrotic lymphadenitis, and recurrent urinary tract infections and pneumonia despite years of IVIG therapy. She had near-normal total IgG, absent IgA and IgE, and protective vaccine antibody titers (pneumococcus, diphtheria, and tetanus) prior to the start of IVIG. Immunophenotyping demonstrated quantitatively normal number of total B cells, absent CD27+ memory B cells, and normal T cell subsets. She had reassuring lymphocyte proliferation with mitogen stimulation and normal TLR function. Dihydrorhodamine (DHR) flow cytometric assay demonstrated abnormally low neutrophil oxidative burst activity. Gene sequencing revealed homozygous nonsense mutation in the NCF-1 gene (c.75-76del; pY26Hfs*26) and a pathogenic heterozygous variant in FANCM (c.3732-3735dup; p. Leu1246Asnfs*8). She was started on daily prophylactic trimethoprim-sulfamethoxazole and itraconazole. She has marked decrease in infections/hospitalizations and her lupus nephritis is well controlled with hydroxy-chloroquine. She is being referred to the NIH for HSCT evaluation. The majority of CGD cases in the United States are due to X-linked CGD (70%) and AR variants in NCF1 (20%). While rare, discoid lupus erythematosus and SLE have been documented with X-linked forms of CGD. Similarly, there are few case reports of antibody deficiency in patients with CGD. This is the first documented case of AR CGD with SLE/lupus nephritis and humoral immune defects. Additionally, we report a disease-causing, novel variant in the NCF1 gene. This case report adds to the existing literature on CGD and associated diseases.

Effect of remote ischemic conditioning on the immune-inflammatory profile in patients with traumatic hemorrhagic shock in a randomized controlled trial

Resuscitation induced ischemia/reperfusion predisposes trauma patients to systemic inflammation and organ dysfunction. We investigated the effect of remote ischemic conditioning (RIC), a treatment shown to prevent ischemia/reperfusion injury in experimental models of hemorrhagic shock/resuscitation, on the systemic immune-inflammatory profile in trauma patients in a randomized trial. We conducted a prospective, single-centre, double-blind, randomized, controlled trial involving trauma patients sustaining blunt or penetrating trauma in hemorrhagic shock admitted to a Level 1 trauma centre. Patients were randomized to receive RIC (four cycles of 5-min pressure cuff inflation at 250 mmHg and deflation on the thigh) or a Sham intervention. The primary outcomes were neutrophil oxidative burst activity, cellular adhesion molecule expression, and plasma levels of myeloperoxidase, cytokines and chemokines in peripheral blood samples, drawn at admission (pre-intervention), 1 h, 3 h, and 24 h post-admission. Secondary outcomes included ventilator, ICU and hospital free days, incidence of nosocomial infections, 24 h and 28 day mortality. 50 eligible patients were randomized; of which 21 in the Sham group and 18 in the RIC group were included in the full analysis. No treatment effect was observed between Sham and RIC groups for neutrophil oxidative burst activity, adhesion molecule expression, and plasma levels of myeloperoxidase and cytokines. RIC prevented significant increases in Th2 chemokines TARC/CCL17 (P < 0.01) and MDC/CCL22 (P < 0.05) at 24 h post-intervention in comparison to the Sham group. Secondary clinical outcomes were not different between groups. No adverse events in relation to the RIC intervention were observed. Administration of RIC was safe and did not adversely affect clinical outcomes. While trauma itself modified several immunoregulatory markers, RIC failed to alter expression of the majority of markers. However, RIC may influence Th2 chemokine expression in the post resuscitation period. Further investigation into the immunomodulatory effects of RIC in traumatic injuries and their impact on clinical outcomes is warranted.ClinicalTrials.gov number: NCT02071290.

Fpr2-/- Mice Developed Exacerbated Alcohol-Associated Liver Disease.

Alcohol-associated liver disease (ALD) is the most common chronic liver disease and carries a significant healthcare burden. ALD has no long-term treatment options aside from abstinence, and the mechanisms that contribute to its pathogenesis are not fully understood. This study aimed to investigate the role of formyl peptide receptor 2 (FPR2), a receptor for immunomodulatory signals, in the pathogenesis of ALD. WT and Fpr2-/- mice were exposed to chronic-binge ethanol administration and subsequently assessed for liver injury, inflammation, and markers of regeneration. The differentiation capacity of liver macrophages and the oxidative burst activity of neutrophils were also examined. Compared to WT, Fpr2-/- mice developed more severe liver injury and inflammation and had compromised liver regeneration in response to ethanol administration. Fpr2-/- mice had fewer hepatic monocyte-derived restorative macrophages, and neutrophils isolated from Fpr2-/- mice had diminished oxidative burst capacity. Fpr2-/- MoMF differentiation was restored when co-cultured with WT neutrophils. Loss of FPR2 led to exacerbated liver damage via multiple mechanisms, including abnormal immune responses, indicating the crucial role of FPR2 in ALD pathogenesis.

Phagocytic and oxidative burst activity of neutrophils in type 2 diabetic patients with foot ulcers

Introduction and Aim: Diabetic foot ulcers are common complications seen in diabetic patients. Treatment of this disabling foot sore remains a challenge to health care professionals. This study aimed at evaluating whether the neutrophils from type 2 diabetic patients with foot ulcers present an impairment of phagocytic index and impairment in respiratory burst. We also aimed at understanding whether the impairment in neutrophil phagocytic activity can be alleviated with short course of standard treatment regime for foot ulcers. Methodology: For this case-controled study, 43 participants with type 2 diabetes (18 with foot ulcers and 25 without foot ulcers) were prospectively recruited along with 18 healthy volunteers. Phagocytic activity of neutrophils and respiratory burst of neutrophils was assessed along with ESR, percentage neutrophil counts before and after 2 weeks of standard treatment for foot ulcers. Results: Neutrophils of type 2 diabetic patients (with and without foot ulcers ) showed lower levels of phagocytic index and lower percentage of respiratory burst on comparison with non-diabetic subjects. Furthermore, on receiving treatment for foot ulcers, a significant improvement in neutrophil phagocytic indices were observed, along with improvement in wound ulcer score. Conclusion: Phagocytic activity of the neutrophils is impaired in type 2 diabetics (with and without foot ulcers). Neutrophil phagocytic indices can be improved on glycemic control. Additionally, improvement in neutrophil phagocytic indices after short course treatment for foot ulcers can be useful markers to predict treatment efficacy and in prognosis of diabetic foot ulcers.

Effects of injectable trace element and vitamin supplementation during the gestational, peri-parturient, or early lactational periods on neutrophil functions and pregnancy rate in dairy cows

The aim of this study was to evaluate effects of injectable trace element and vitamin combination on phagocytic, oxidative burst activity of neutrophils and reproductive outcomes in dairy cows. Cows were to assigned to the following groups: (1) injectable trace element supplementation (ITES, n = 44, containing zinc, manganese, copper, selenium); (2) injectable vitamin supplementation (VIT, n = 48, containing vitamins A, D3, E); (3) ITES + VIT (n = 46); and (4) control (CON, n = 44). Cows were administered four injections between 230 and 260 days of the gestational period, on day of parturition, and 30 days postpartum. Neutrophil function was assessed at 10 days before and after calving. Phagocytosis was greater in cows of the ITES + VIT group at 10 days prepartum (P < 0.05) while oxidative burst was similar among groups. There were greater non-esterified fatty acid (NEFA) concentrations in cows of the ITES+VIT group at 10 days prepartum (P < 0.05). Cows supplemented with ITES+ VIT had less SOD activity than those supplemented with ITES or vitamin during the pre- to post-partum transition period (P < 0.05). The total odds of pregnancy were greater in cows supplemented with trace element and/or vitamin (P < 0.05). In conclusion, supplementation of ITES and/or VIT resulted in an increased total pregnancy rate. Vitamin or trace element supplementation did not differ with the control group in both the prepartum and postpartum period for immune variables. There, however, was greater phagocytosis in cows supplemented with vitamin and trace elements during the prepartum period that might be related to metabolic-induced inflammation.

Vitamin C and Neutrophil Function: Findings from Randomized Controlled Trials.

Vitamin C is known to support immune function and is accumulated by neutrophils to millimolar intracellular concentrations suggesting an important role for the vitamin in these cells. In this review, the effects of vitamin C, as a mono- or multi-supplement therapy, on neutrophil function were assessed by conducting a systematic review of randomized controlled trials (RCTs). Specifically, trials which assessed neutrophil migration (chemotaxis), phagocytosis, oxidative burst, enzyme activity, or cell death (apoptosis) as primary or secondary outcomes were assessed. A systematic literature search was conducted using the Cochrane Central Register of Controlled Trials, EMBASE, Embase Classic, Joanna Briggs Institute EBP, Ovid MEDLINE®, Ovid MEDLINE® In-Process & Other Non-Indexed Citations, Ovid Nursing Database, CINAHL and PubMed database, which identified 16 eligible RCTs. Quality appraisal of the included studies was carried out using the Cochrane Risk of Bias tool. Three of the studies assessed neutrophil chemotaxis in hospitalised patients or outpatients, two of which showed improved neutrophil function following intravenous vitamin C administration. Ten RCTs assessed neutrophil phagocytosis and/or oxidative burst activity; five were exercise studies, one in smokers, one in myocardial infarction patients and three in healthy volunteers. Two of the multi-supplement studies showed a difference between the intervention and control groups: increased oxidative burst activity in athletes post-exercise and decreased oxidant generation in myocardial infarction patients. Two studies assessed neutrophil enzyme activity; one showed deceased antioxidant enzyme activity in divers and the other showed increased antioxidant enzyme activity in athletes. One final study showed decreased neutrophil apoptosis in septic surgical patients following intravenous vitamin C administration. Overall, 44% of the RCTs assessed in this review showed effects of vitamin C supplementation on neutrophil functions. However, the studies were very heterogeneous, comprising different participant cohorts and different dosing regimens. There were also a number of limitations inherent in the design of many of these RCTs. Future RCTs should incorporate prescreening of potential participants for low vitamin C status or utilize cohorts known to have low vitamin status, such as hospitalized patients, and should also comprise appropriate vitamin C dosing for the cohort under investigation.

Hazardous effect of the maintenance dose of iron sucrose versus iron dextran on oxidative burst activity of neutrophils in patients on regular hemodialysis; a pilot study

Introduction: Intravenous (IV) iron which is a potent pro-oxidant may decrease the phagocytic function of neutrophils and subsequently leads to repeated bacterial infections in patients on chronic regular hemodialysis (HD).Objectives: We aimed to evaluate the effect of IV iron dextran versus iron sucrose on oxidative burst activity of neutrophils in HD patients.Patients and Methods: A crossover study was included 20 prevalent HD patients were randomly divided into two groups; 10 patients each. Each group received a single dose of 100 mg iron sucrose or iron dextran then shifted to the other type after 4 weeks as a washout period. Patients with evidence of acute infections, diabetes mellitus, chronic liver disease, active collagen disease or any other acquired immune deficiency diseases were excluded. Patients on immunosuppressive drugs or with hypersensitivity to iron therapy or evidence of iron overload (TSAT &gt; 50% and/or ferritin &gt; 800 ng/mL) were also excluded. Erythropoietin maintenance dose was given to all patients. Urea reduction ratio (URR %), C-reactive protein (CRP) titer, complete blood picture and iron study were conducted. Neutrophil oxidative burst test was performed by flow-cytometry with an estimation of stimulation index before and after IV iron dosage. Results: Twenty patients (9 males, 11 females; mean age 49.40 ± 9.02 years) on chronic HD for a mean time of 10 ± 7.32 years. After 100 mg of IV iron infusion in HD patients, a highly significant difference in oxidative burst test of neutrophil cells before and after administration of iron regardless of its type was detected (P &lt; 0.01). This change over time is not significantly different between two types of iron (P &gt; 0.05). However, a significant increase in CRP titer after administration of iron dextran was seen (P = 0.016). Conclusion: Maintenance dosage of either IV iron sucrose or iron dextran (100 mg) had a hazardous effect on the neutrophilic phagocytosis demonstrated by the significant rise in the oxidative burst index in HD patients.

Amaranth (Amaranthus cruentus L.) and canola (Brassica napus L.) oil impact on the oxidative metabolism of neutrophils in the obese patients*

Context: Amaranth and canola oils have been used traditionally. Amaranth has been identified as being of interest because of its outstanding nutritive value. Amaranth oil is a rich source of highly unsaturated fats and so could be a valuable dietary alternative for individuals affected with obesity. Reactive oxygen species (ROS) are postulated to be involved in systemic inflammation and oxidative stress. Activated polymorphonuclear neutrophils (PMNs) generate high amounts of reactive oxygen species.Objective: Our study investigates the impact of amaranth and canola oils supplementation on oxidative metabolism in patients with obesity. We hypothesized that, due to its lipid-lowering and antioxidant properties, amaranth and canola oil would protect against oxidative stress.Materials and methods: We tested 19 obese patients [body mass index (BMI) = 41.1 ± 7.8 kg/m2, (mean ± SD)]. The protocol consisted of two stages: a run-in phase of 2 weeks and an experimental stage – canola or amaranth oil supplementation (20 mL/d) with calorie restriction diet for 3 weeks. The neutrophil oxidative burst was expressed by fluorescence intensity (IF).Results: The oxidative burst had increased significantly at the end of treatment in both groups IF: (21.4 ± 11.15 vs. 35.9 ± 20.3; mean ± SD) p < 0.05. The levels of IF were significantly higher in neutrophils of patients who received canola oil (41.05 ± 25.3) compared to those who received amaranth oil (28.4 ± 11.8) p < 0.05.Conclusions: Canola oil exerts possible effects on oxidative burst activity in neutrophils in vivo conditions.

Phagocytic and oxidative burst activity of phagocytic cells in peripheral blood and uterine washings in cows with clinical endometritis before and after intrauterine use of cephapirin and methisoprinol.

The aim of the study was to evaluate phagocytic and killing activity of phagocytic cells in blood and uterine flush of cows with endometritis before and after intrauterine (i.u.) administration of cephapirin and methisoprinol. The research was carried out on 28 cows with clinical endometritis. Animals were divided into four groups, each composed of seven cows, depending on the i.u. treatment used: Group A-cephapirin; Group B-methisoprinol; Group C-cephapirin and methisoprinol at the same time; and a control group-without medication. Using flow cytometry technique, the phagocytic activity of granulocytes and monocytes was identified, as well as the oxidative burst activity of neutrophils in the peripheral blood and uterine washings. Summarizing the results of the research, i.u. infusion of cephapirin caused a reduction in the phagocytic and killing activity of phagocytes. The i.u. use of methisoprinol increased phagocytic and killing activity of phagocytes in the uterus. Administering both listed substances simultaneously showed a decrease in phagocytosis, presumably due to the dominating inhibitor effect of the antibiotic. However, also an increase of mean fluorescence intensity was observed, presumably caused by the methisoprinol. Intrauterine use of immunostimulatory substances, can improve the effectiveness of the treatment of endometritis in cows.

Effects of prepartum dietary cation-anion difference and source of vitamin D in dairy cows: Health and reproductive responses

The objectives of the experiment were to evaluate the effects of feeding diets with distinct dietary cation-anion difference (DCAD) levels and supplemented with 2 sources of vitamin D during the prepartum transition period on postpartum health and reproduction in dairy cows. The hypotheses were that feeding acidogenic diets prepartum would reduce the risk of hypocalcemia and other diseases, and the benefits of a negative DCAD treatment on health would be potentiated by supplementing calcidiol compared with cholecalciferol. Cows at 252 d of gestation were blocked by parity (28 nulliparous and 52 parous cows) and milk yield within parous cows, and randomly assigned to 1 of 4 treatments arranged as a 2 × 2 factorial, with 2 levels of DCAD, positive (+130 mEq/kg) or negative (-130 mEq/kg), and 2 sources of vitamin D, cholecalciferol or calcidiol, fed at 3 mg for each 11 kg of diet dry matter. The resulting treatment combinations were positive DCAD with cholecalciferol (PCH), positive DCAD with calcidiol (PCA), negative DCAD with cholecalciferol (NCH), and negative DCAD with calcidiol (NCA), which were fed from 252 d of gestation to calving. After calving, cows were fed the same lactation diet supplemented with cholecalciferol at 0.70 mg for every 20 kg of dry matter. Blood was sampled 7 d before parturition, and at 2 and 7 d postpartum to evaluate cell counts and measures of neutrophil function. Postpartum clinical and subclinical diseases and reproductive responses were evaluated. Feeding a diet with negative DCAD eliminated clinical hypocalcemia (23.1 vs. 0%) and drastically reduced the incidence and daily risk of subclinical hypocalcemia, and these effects were observed in the first 48 to 72 h after calving. The diet with negative DCAD tended to improve the intensity of oxidative burst activity of neutrophils in all cows prepartum and increased the intensity of phagocytosis in parous cows prepartum and the proportion of neutrophils with killing activity in parous cows postpartum (58.5 vs. 67.6%). Feeding calcidiol improved the proportion of neutrophils with oxidative burst activity (60.0 vs. 68.7%), reduced the incidences of retained placenta (30.8 vs. 2.5%) and metritis (46.2 vs. 23.1%), and reduced the proportion of cows with multiple diseases in early lactation. Combining the negative DCAD diet with calcidiol reduced morbidity by at least 60% compared with any of the other treatments. Cows with morbidity had lower blood ionized Ca and serum total Ca concentrations than healthy cows. Treatments did not affect the daily risk of hyperketonemia in the first 30 d of lactation. Despite the changes in cow health, manipulating the prepartum DCAD did not influence reproduction, but feeding calcidiol tended to increase the rate of pregnancy by 55%, which reduced the median days open by 19. In conclusion, feeding prepartum cows with a diet containing a negative DCAD combined with 3 mg of calcidiol benefited health in early lactation.

Short communication: Relationship between methods for measurement of serum electrolytes and the relationship between ionized and total calcium and neutrophil oxidative burst activity in early postpartum dairy cows

The objectives of this study were to (1) compare a test for serum measurement of total Ca (tCa), Mg, and P (VetTest Chemistry Analyzer, IDEXX Laboratories Inc., Westbrook, ME) to reference methods (spectrophotometric assays on a Beckman Coulter 640e automated clinical chemistry analyzer; Beckman Coulter, Brea, CA), (2) determine the relationship between ionized Ca (iCa) and reference method tCa in the immediate postpartum period, and (3) assess the relative value of these blood Ca indices as predictors of neutrophil oxidative burst activity. Samples were collected from multiparous Holstein cows (n = 33) over the first 5 d in milk. A total of 183 samples for objective 1 and 181 samples for objective 2 were available. Neutrophil oxidative burst activity was assessed once between 2 and 5 d in milk (n = 29). Linear regression demonstrated strong relationships between serum tCa, Mg, and P concentrations measured by the VetTest compared with the reference method. Bland Altman analysis indicated that the VetTest values were higher than the reference method by 0.22 mmol/L for tCa, 0.12 mmol/L for Mg, and 0.16 mmol/L for P. Compared with hypocalcemia categorized at ≤2.0 or ≤2.125 mmol/L with the reference method tCa, thresholds for the VetTest measured tCa of ≤2.23 mmol/L (sensitivity = 87%, specificity = 89%) or ≤2.30 mmol/L (sensitivity = 86%, specificity = 96%) could be used. The relationship between whole-blood iCa and reference method serum tCa differed by sampling time point after calving. Compared with identification of hypocalcemia with serum tCa measurements from the reference method (thresholds of ≤2.0 and 2.125 mmol/L), a whole-blood iCa threshold of ≤1.17 mmol/L resulted in the highest combined sensitivities (94 and 82%) and specificities (80 and 94%) at either threshold. Ionized Ca measurements were more consistently related to outcomes of neutrophil oxidative burst activity measured in vitro. The VetTest measurements of serum tCa reliably identified hypocalcemia when thresholds were adjusted to account for the bias of the test. The variation in the relationship between iCa and reference method tCa in the days following parturition suggest that these measures cannot be used interchangeably as indicators of Ca status. The more consistent associations between iCa and in vitro measures of neutrophil function, compared with tCa, indicated that this may be a more sensitive predictor of functional outcomes associated with postpartum Ca status.

Individuals with obesity and type 2 diabetes have additional immune dysfunction compared with obese individuals who are metabolically healthy

ObjectiveThe objective of the current study was to compare the responses to different ex vivo immunogenic challenges between immune cells derived from metabolically healthy subjects with obesity and subjects with...

Supplementation with rumen-protected methionine or choline during the transition period influences whole-blood immune response in periparturient dairy cows

Methionine, together with Lys, is the most limiting AA for milk production in dairy cows. Besides its crucial role in milk production, Met and its derivate metabolites (e.g., glutathione, taurine, polyamines) are well-known immunonutrients in nonruminants, helping support and boost immune function and activity. In the present study, the effects of Met or choline, as its precursor, were investigated using an ex vivo whole blood challenge. The study involved 33 multiparous Holstein cows (from a larger cohort with a factorial arrangement of treatments) assigned from d -21 to +30 relative to parturition to a basal control (CON) diet, CON plus rumen-protected Met (MET, Smartamine M, Adisseo NA, Alpharetta, GA) at a rate of 0.08% of dry matter, or CON plus rumen-protected choline (CHOL, ReaShure, Balchem Inc., New Hampton, NY) at 60 g/d. Blood was sampled on d -15, -7, 2, 7, and 20 for ex vivo lipopolysaccharide (LPS) challenge, and on d 1, 4, 14, and 28 relative to parturition for phagocytosis and oxidative burst assays. The MET cows had greater energy-corrected milk production and milk protein content. Overall, IL-6 response to LPS increased around parturition, whereas IL-1β remained constant, casting doubt on the existence of systemic immunosuppression in the peripartal period. Supplementation with MET dampened the postpartal blood response to LPS (lower IL-1β), while improving postpartum neutrophil and monocyte phagocytosis capacity and oxidative burst activity. In contrast, CHOL supplementation increased monocyte phagocytosis capacity. Overall, the data revealed a peripartal immune hyper-response, which appeared to have been mitigated by MET supplementation. Both MET and CHOL effectively improved immune function; however, MET affected the immune and antioxidant status before parturition, which might have been beneficial to prepare the cow to respond to metabolic challenges after parturition. These results provide insights on potential differences in the immunomodulatory action of methionine and choline in dairy cows. As such, the effects observed could have implications for ration formulation and dietary strategies.

Impairment of neutrophil oxidative burst in children with sickle cell disease is associated with heme oxygenase-1.

Sickle cell disease is a risk factor for invasive bacterial infections, and splenic dysfunction is believed to be the main underlying cause. We have previously shown that the liberation of heme in acute hemolysis can induce heme oxygenase-1 during granulopoiesis, impairing the ability of developing neutrophils to mount a bactericidal oxidative burst, and increasing susceptibility to bacterial infection. We hypothesized that this may also occur with the chronic hemolysis of sickle cell disease, potentially contributing to susceptibility to infections. We found that neutrophil oxidative burst activity was significantly lower in treatment-naïve children with sickle cell disease compared to age-, gender- and ethnicity-matched controls, whilst degranulation was similar. The defect in neutrophil oxidative burst was quantitatively related to both systemic heme oxygenase-1 activity (assessed by carboxyhemoglobin concentration) and neutrophil mobilization. A distinct population of heme oxygenase-1-expressing cells was present in the bone marrow of children with sickle cell disease, but not in healthy children, with a surface marker profile consistent with neutrophil progenitors (CD49d(Hi) CD24(Lo) CD15(Int) CD16(Int) CD11b(+/-)). Incubation of promyelocytic HL-60 cells with the heme oxygenase-1 substrate and inducer, hemin, demonstrated that heme oxygenase-1 induction during neutrophilic differentiation could reduce oxidative burst capacity. These findings indicate that impairment of neutrophil oxidative burst activity in sickle cell disease is associated with hemolysis and heme oxygenase-1 expression. Neutrophil dysfunction might contribute to risk of infection in sickle cell disease, and measurement of neutrophil oxidative burst might be used to identify patients at greatest risk of infection, who might benefit from enhanced prophylaxis.

Systemic lupus erythematosus onset in lupus-prone B6.MRL/lpr mice Is influenced by weight gain and Is preceded by an increase in neutrophil oxidative burst activity

In this study, we assessed whether weight gain influenced the systemic lupus erythematosus (SLE) onset and/or outcome, and examined the role that reactive oxygen species (ROS) production by neutrophils played in the SLE onset and/or outcome. Female control (C57BL/6) and lupus-prone B6.MRL/lpr mice (CM and LPM, respectively) at 4 weeks old were fed standard diet or standard diet plus cafeteria diet during 12 weeks. SLE diagnosis relied on the presence of both antinuclear antibodies (ANA) and renal abnormalities. We found that the percentage of weight gain in CM and LPM increased as a function of the length of cafeteria diet feeding period, but it was not associated with energy intake. Cafeteria diet-fed CM and LPM at 8 and 12 weeks old were overweight, while CM and LPM at 16 weeks old were obese. Compared with standard diet-fed CM and LPM, cafeteria diet-fed CM and LPM exhibited elevated glucose and total cholesterol levels, and diminished triglycerides levels. Standard diet-fed 16-week-old LPM and cafeteria diet-fed 12-week-old LPM had nephritis, characterized by the increased interstitial infiltration of leukocytes. Cafeteria diet-induced weight gain rose the frequency of homogeneous and speckled ANA staining patterns in the 12- and 16-week-old LPM groups. Together, these results indicated that weight gain anticipated the SLE onset. In addition, neutrophils from cafeteria diet-fed 8-week-old LPM exhibited augmented ROS production capacity; in standard diet-fed LPM, such rise occurred only in the 16-week-old group. Thus, the neutrophil ROS production capacity was increased before the SLE onset and during its outcome. Overweight and obese CM and LPM displayed elevated levels of kidney, liver, heart, and spleen lipid peroxidation. In conclusion, cafeteria diet-induced weight gain is associated with the increased production of ANA and neutrophil-derived ROS, which may contribute to accelerate the SLE onset.

Neutrophil function in healthy aged horses and horses with pituitary dysfunction

Immunosuppression leading to opportunist bacterial infection is a well-recognized sequela of equine pituitary pars intermedia dysfunction (PPID). The mechanisms responsible for immune dysfunction in PPID however, are as of yet poorly characterized. Horses with PPID have high concentrations of hormones known to impact immune function including α-melanocyte stimulating hormone (α-MSH) and insulin. α-MSH and related melanocortins have been shown in rodents and people to impair neutrophil function by decreasing superoxide production (known as oxidative burst activity), migration and adhesion. The goal of this study was to determine if neutrophil function is impaired in horses with PPID and, if so, to determine if plasma α-MSH or insulin concentration correlated with the severity of neutrophil dysfunction. Specifically, neutrophil phagocytosis, oxidative burst activity, chemotaxis and adhesion were assessed. Results of this study indicate that horses with PPID have reduced neutrophil function, characterized by decreased oxidative burst activity and adhesion. In addition, chemotaxis was greater in healthy aged horses than in young horses or aged horses with PPID. Plasma insulin: α-MSH ratio, but not individual hormone concentration was correlated to neutrophil oxidative burst activity. In summary, neutrophil function is impaired in horses with PPID, likely due to altered hormone concentrations and may contribute to increased risk of opportunistic infections. Whether regulation of hormone concentration profiles in horses with PPID using therapeutic intervention improves neutrophil function and reduces infections needs to be explored.

In vitro interferon γ improves the oxidative burst activity of neutrophils in patients with chronic granulomatous disease with a subtype of gp91phox deficiency.

Aim of this studyChronic granulomatous disease (CGD) is a genetically heterogeneous primary immunodeficiency caused by a defect in phagocyte production of oxygen metabolites, and resulting in infections produced by catalase-positive microorganisms and fungi. Interferon γ (IFN-γ) has a multitude of effects on the immune system. Although preliminary studies with CGD patients on treatment with IFN-γ showed that it enhanced phagocytosis and superoxide production, ongoing studies did not reveal a significant increase of this function. Here we investigated the oxidative capacity of phagocytes in different subtypes of CGD patients on treatment with IFN-γ in vitro.Material and methodsFifty-seven patients with CGD from 14 immunology centres were enrolled to our multi-centre study. Twenty-one patients were studied as controls. Oxidative burst assay with dihydrorhodamine 123 (DHR) was used and the stimulation index (SI) was calculated with respect to CGD subtypes in both neutrophils and monocytes before, and then one and 24 hours after adding IFN-γ.ResultsUpon comparison of the SIs of the patients’ neutrophils before in vitro IFN-γ at hour 0, and after adding IFN-γ at hour 1 and 24 were compared, and the differences were determined between hours 0-24 and hours 1-24. This difference was especially apparent between hours 1-24. In CGD subtypes, particularly in gp91phox subtype, it was seen that, following in vitro IFN-γ, SIs of neutrophils began to increase after hour 1, and that increase became more apparent at hour 24.ConclusionsOur study showed that IFN-γ treatment may increase the oxidative bursting activity by increasing the superoxide production in neutrophils, particularly in gp91phox subtype.

Is it X-Linked or autosomal recessive chronic granulomatous disease?

Background Neutrophil oxidative burst (NOB) activity as assessed by dihydrorhodamine-123 flow cytometric assay, is used in the diagnosis of chronic granulomatous disease (CGD) and may provide insight into its inheritance pattern. Case report We present a consanguineous family with complete absence of NOB activity in three male siblings. Each affected sibling suffered a severe form of CGD characterised by clinical manifestations within the first year of life, recurrent bacterial and fungal pneumonia, and death in two siblings by the ages of 3 and 4. This complete absence of NOB activity and severe phenotype is atypical for classic autosomal recessive CGD. The unaffected parents and siblings displayed normal NOB activity. In addition, no bimodal peak was detected in the mother, which is atypical for X-linked CGD. Extreme skewing of lyonisation in the maternal X-chromosome was excluded. CGD mutation analysis was performed by GeneDx (Gaithersburg, USA, 2008) on the proband. A novel autosomal recessive p67-phox (NCF2) homozygous deletion mutation [NM_000433.3(NCF2): c.482delA] was identified. Objective and results A targeted bi-directional PCR sequencing analysis was developed in a local molecular laboratory (Pathology Queensland) to help identify this novel mutation in other members of this family. The heterozygous carrier status in both parents and the homozygous mutation in a newborn male sibling were demonstrated by this analysis. Discussion This case report confirms the autosomal recessive mode of inheritance of this novel c.482delA mutation in the p67-phox (NCF2) gene. The mutation is predicted to result in a frame shift and premature stop codon [NP_000424.2(NCF2): p.Lysl61 Argfs*16], potentially explaining the complete absence of NOB activity and severe clinical phenotype in the affected male siblings. Neutrophil oxidative burst (NOB) activity as assessed by dihydrorhodamine-123 flow cytometric assay, is used in the diagnosis of chronic granulomatous disease (CGD) and may provide insight into its inheritance pattern. We present a consanguineous family with complete absence of NOB activity in three male siblings. Each affected sibling suffered a severe form of CGD characterised by clinical manifestations within the first year of life, recurrent bacterial and fungal pneumonia, and death in two siblings by the ages of 3 and 4. This complete absence of NOB activity and severe phenotype is atypical for classic autosomal recessive CGD. The unaffected parents and siblings displayed normal NOB activity. In addition, no bimodal peak was detected in the mother, which is atypical for X-linked CGD. Extreme skewing of lyonisation in the maternal X-chromosome was excluded. CGD mutation analysis was performed by GeneDx (Gaithersburg, USA, 2008) on the proband. A novel autosomal recessive p67-phox (NCF2) homozygous deletion mutation [NM_000433.3(NCF2): c.482delA] was identified. A targeted bi-directional PCR sequencing analysis was developed in a local molecular laboratory (Pathology Queensland) to help identify this novel mutation in other members of this family. The heterozygous carrier status in both parents and the homozygous mutation in a newborn male sibling were demonstrated by this analysis. This case report confirms the autosomal recessive mode of inheritance of this novel c.482delA mutation in the p67-phox (NCF2) gene. The mutation is predicted to result in a frame shift and premature stop codon [NP_000424.2(NCF2): p.Lysl61 Argfs*16], potentially explaining the complete absence of NOB activity and severe clinical phenotype in the affected male siblings.

Deficient cytokine expression and neutrophil oxidative burst contribute to impaired cutaneous wound healing in diabetic, biofilm‐containing chronic wounds

Diabetic patients exhibit dysregulated inflammatory and immune responses that predispose them to chronic wound infections and the threat of limb loss. The molecular underpinnings responsible for this have not been well elucidated, particularly in the setting of wound biofilms. This study evaluates host responses in biofilm-impaired wounds using the TallyHo mouse, a clinically relevant polygenic model of type 2 diabetes. No differences in cytokine or Toll-like receptor (TLR) expression were noted in unwounded skin or noninoculated wounds of diabetic and wild-type mice. However, diabetic biofilm-containing wounds had significantly less TLR 2, TLR 4, interleukin-1β, and tumor necrosis factor-α expression than wild-type wounds with biofilm (all p < 0.001). Both groups had similar bacterial burden and neutrophil infiltration after development of biofilms at 3 days postwounding, but diabetic wounds had significantly less neutrophil oxidative burst activity. This translated into a log-fold greater bacterial burden and significant delay of wound epithelization for biofilm-impaired diabetic wounds at 10 days postwounding. These results suggest that impaired recognition of bacterial infection via the TLR pathway leading to inadequate cytokine stimulation of antimicrobial host responses may represent a potential mechanism underlying diabetic susceptibility to wound infection and ulceration.